Promoting microbiology education through the iGEM synthetic biology competition.

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education.

The ECF sigma factor RpoI of R. leguminosarum initiates transcription of the vbsGSO and vbsADL siderophore biosynthetic genes in vitro.

When complexed with Escherichia coli RNA polymerase core enzyme, purified RpoI protein of Rhizobium leguminosarum initiated transcription in vitro from promoters of the vbsADL and vbsGSO operons, which are needed to synthesise the siderophore vicibactin. There is a single transcription initiation site for rpoI, regardless of whether the cells are grown in Fe-replete or Fe-depleted media, but levels of rpoI mRNA were reduced, though not abolished, in the presence of Fe. Unlike PvdS, a similar Pseudomonas sigma factor needed to transcribe genes involved in pyoverdine synthesis, RpoI transcribes vbsADL and vbsGSO in the absence of the cognate siderophore.

RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum.

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Fe-mediated gene regulation.

Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains.

A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells.

Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases.

A novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified. The homology was greatest to the GST enzymes of higher plants. The Rhizobium gstA gene was normally expressed at a very low level.

High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators.

The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective.